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The primary purpose of this study was to identify if serum 25-hydroxyvitamin D (25 (OH) D) concentrations predict muscular weakness after intense exercise. We hypothesized that pre-exercise serum 25 (OH) D concentrations inversely predict exercise-induced muscular weakness. Fourteen recreationally active adults participated in this study. Each subject had one leg randomly assigned as a control. The other leg performed an intense exercise protocol. Single-leg peak isometric force and blood 25 (OH) D, aspartate and alanine aminotransferases, albumin, interferon (IFN)-γ, and interleukin-4 were measured prior to and following intense exercise. Following exercise, serum 25 (OH) D concentrations increased (p< 0.05) immediately, but within minutes, subsequently decreased (p< 0.05). Circulating albumin increases predicted (p< 0.005) serum 25 (OH) D increases, while IFN-γ increases predicted (p< 0.001) serum 25 (OH) D decreases. Muscular weakness persisted within the exercise leg (p< 0.05) and compared to the control leg (p< 0.05) after the exercise protocol. Serum 25 (OH) D concentrations inversely predicted (p< 0.05) muscular weakness (ie, control leg vs. exercise leg peak isometric force) immediately and days (ie, 48-h and 72-h) after exercise, suggesting the attenuation of exercise-induced muscular weakness with increasing serum 25 (OH) D prior to exercise. Based on these data, we conclude that pre-exercise serum 25 (OH) D concentrations could influence the recovery of skeletal muscle strength after an acute bout of intense exercise.